Many hydrophobic peptides from plants are known to be highly biologically active materials with wide ranges of therapeutic applications. For example, asterins are cyclopentapeptides isolated from Aster tartaricus (also known as tartarian aster), known to be potent anti-tumour compounds. Cyclic heptapeptides extracted from Stellaria yunnanensis (also known as chickweed), commonly referred to as yunnanins, have cytotoxic effects on P388 leukemia cells. Likewise, Rubia akane (also known as asian madder) and Rubia cordifolia (also known as indian madder) produce bicyclic hexapeptides with similar cytotoxic activities. Segetalin A is a cyclic hexapeptide from Vaccaria segitalis (also known as Wang Bu Liu Xing or cow cockle) known to have estrogen-like activity. Pseudostellaria heterophylla (also known as Tai Zi Shen, false starwort or Prince Seng) is used as the “ginseng of the lungs.” P. heterophylla is known to contain the cyclic heptapeptide Pseudostellarin D, a well-known Chinese traditional medicine that is used to tonify the “qi” and generate “yin” fluids. The use of Pseudostellarin D as a lung and spleen tonic stems from its inhibition of tyrosinase. In addition, cyclolinopeptides from flax seed are active in suppressing a wide range of immunological responses, including: (i) delayed-type hypersensitivity response; (ii) skin allograph rejection; (iii) graft versus host reaction; (iv) post-adjuvant arthritis; and (v) haemolytic anemia of New Zealand black (NZB) mice.
Potential applications of hydrophobic peptides include, but are not limited to, their use in drug forms and in vaccine formulations. Expression of the genes encoding the cyclolinopeptides is associated with programmed cell death in times of injury to a cell. Accordingly, the recovery and purification of such hydrophobic peptides is important. To date, reported methods and processes for recovery of hydrophobic peptides are cumbersome and do not result in a significant yield of hydrophobic peptides. Moreover, prior inventions involve the extraction of all hydrophobic peptides as a group using silica gel, followed by chromatographic separation in order to isolate individual hydrophobic peptides, which can be problematic and expensive.
Kaufman et al. (Uber ein Oligopeptid aus Leinsamen Chem Ber, 1959, 92: 2805-9) describe a method of recovery of hydrophobic peptides from a precipitated “slime” obtained in the processing of flax seed. Morita et al. (Bioorganic & Medicinal Chemistry Letters, 1997, 7(10): 1269-1272; Tetrahedron, 2002, 58: 5135-5140; Phytochemistry, 2001, 57: 251-260; Tetrahedron, 1999, 55: 967-976) describe the preparation of small amounts of hydrophobic peptides from flax seed, press cake and roots. Their extraction process requires significant quantities of flax material (30 kilograms) extracted with four volumes of hot methanol. The solvent is subsequently removed and the entire mixture subjected to chromatography on a polystyrene column (Diaion™ HP-20) and washed with increasing concentrations of methanol. The hydrophobic peptides remain bound to the column until washed with pure methanol. Polar solutes can be removed from the column in the presence of water. The methanol fraction containing the hydrophobic peptides is then subjected to normal phase silica gel chromatography using a gradient of chloroform and methanol (100:0 to 0:100), wherein the peptides are eluted with relatively low concentrations of methanol. A fraction of the methanol:chloroform gradient is recovered and subjected to reverse-phase chromatography using a methanol and acetonitrile gradient to isolate individual fractions containing individual hydrophobic peptides.
Stefanowicz (Acta Biochimica Polonica, 2001, 48: 1125-1129) disclose recovery of hydrophobic peptides from flax seed. The extraction of cyclolinopeptides is done using 5 grams of ground flax seed, extracted overnight in 100 milliliters of acetone. The acetone fraction is concentrated in vacuo and the resulting mixture dissolved in methanol and hydrolyzed using 10% sodium hydroxide. After drying, the fraction containing the cyclolinopeptides is isolated by ethyl acetate extraction. Such a process does not allow the separation and isolation of each hydrophobic peptide.
A recent publication by Brühl et al. (J. Agric. Food Chem., 2007, 55: 7864-7868) disclose a method for isolating hydrophobic cyclopeptides which provides low yields of product. Bitter linseed oil is prepared from seeds with a laboratory screw press with an 8-mm nozzle at temperatures not exceeding 60° C. and a speed of screw rotation of 35 rpm. The temperature of the freshly obtained oil (30% yield) does not exceed 40° C. No additional steps are taken to maximize oil recovery and peptide extraction from the seeds. An aliquot of the bitter linseed oil is subsequently dissolved in heptane, a solvent not approved for food extraction, and then extracted 3 times with methanol/water solvent (6:4; v/v; 200 mL each). The aqueous layers are filtered through a wet filter paper to separate traces of oil from the layer, and the solvent is then removed in vacuum. A bitter-tasting fraction is obtained from the oil, which is then dissolved in methanol/diethyl ether (1:1; v/v; 1 mL) and placed onto the top of a glass column filled with a slurry of silica gel. Chromatography is performed with solvent mixtures using ratios of diethyl ether and ethanol. The individual fractions are freed from the solvent under vacuum and then taken up in water. Purification of cyclolinopeptide E is accomplished by reverse-phase high-performance liquid chromatography (HPLC). Aliquots of certain fractions thought to contain the bitter-taste compound are dissolved in water/ethanol and separated by HPLC on a column connected to a UVD 340-type US/vis detector operating at a wavelength of 210 nm. Chromatography is performed starting with a mixture of methanol/water (75:25; v/v) and ending with 100% methanol within 25 minutes. The effluent is collected in 1-min fractions using a fraction collector, with each fraction freed from solvent and residues taken up in water upon ultrasonification.
The recovery of hydrophobic peptides from plants is difficult, laborious, and expensive. Chemical synthesis of the peptides has been considered as an alternative source for candidate therapeutic hydrophobic peptides. For example, Wiezorek et al. (Peptide Res, 1991, 4: 275-283) disclose synthetic preparation of cyclolinopeptide A on Merrifield resin using tert-butyloxycarbonyl protected amino acids. The product is split from the resin by a mixture of trifluoro acetic acid and sulphuric acid and then cyclized by Castro's reagent, dissolved in ethyl acetate and subsequently washed with 1N NaOH, 1N HCl and water. The final product is purified by HPLC. However, the product yields of such synthetic methods are also very low, and are not adequate solutions to the difficulties encountered with recovering endogenous peptides.